Vitamin A is needed by the body to regulate expression of numerous genes with diverse cellular functions. The effects of vitamin A on gene expression arise through interactions of all-trans or 9-cis retinoic acid with members of two families of ligand dependent transcription factors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs). All-trans retinoic acid binds only to RARs with high affinity; whereas its 9-cis isomer binds with high affinity to both RARs and RXRs. The actions of all-trans and 9-cis retinoic acid in regulating cellular responses are distinct and not interchangeable. Only recently has convincing data become available regarding the enzymatic formation of all-trans retinoic acid from its precursor retinol and there is still a near complete absence of data regarding how 9-cis retinoic acid is formed within tissues. We have obtained and characterized a full length cDNA clone which, when expressed in CHO cells, avidly catalyzes oxidation of 9-cis retinol to 9-cis retinaldehyde; the first enzymatic step needed for the formation of 9- cis retinoic acid. The CHO cell expressed cDNA does not catalyze all- trans retinol oxidation. Based on this result and other data described in this application, it is clear that we have identified a novel steriospecific enzyme, 9-cis retinol dehydrogenase (9cRDH) which may play an important role in 9-cis retinoic acid formation. This proposal consists of 4 Aims which comprehensively explore the biochemistry and physiology of this novel enzyme. In Aim 1, we will define the biochemical properties of human and murine 9cRDH and generate antibodies to recombinant forms of the proteins. We will employ, in Aim 2, in situ hybridization and immunocytochemical techniques to explore the cellular localization of 9cRDH in adult mouse tissues and its spatial and temporal pattern of distribution in mouse embryos. Using the highly characterized mouse testis system as a model, in Aim 3, relationships between 9cRDH expression pattern and the actions of vitamin A in maintaining normal testis function will be identified. Aim 4 will investigate the physiologic significance of the 9cRDH using the techniques of targeted gene disruption.